A major objective of this research proposal is to contribute to an increased understanding of the transcription of influenza viral RNA and involves an investigation of the cellular factors as well as the viral enzymes involved in the process. A cellular protein(s), F2, was found to stimulate the in vitro rate of transcription resulting in the formation of long cRNA transcripts containing polyadenylate sequences. The possibility that F2 affects initiation will be investigated by examining the 5'-termini of transcripts formed in the presence and absence of this protein. Another role for F2 being considered is that the protein modifies the virion proteins necessary for transcription. A procedure for obtaining the polymerase proteins from ribonucleoprotein-polymerase complexes was developed. This method, it is believed, will permit a study of possible F2 interaction with the polymerase. Identification of the proteins required for RNA-dependent RNA polymerase and the recently detected polyadenylate polymerase, as well as a determination of whether the two activities can be separated. Another major goal is the preparation of a replicative complex capable of synthesizing vRNA. Experiments to determine the time of maximum vRNA synthesis by infected cells will first be carried out. After the appropriate time is determined, cell-free extracts will be prepared and tested for their ability to synthesize vRNA. The extracts will then be fractionated in an effort to isolate the replicative complex.